This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this R&D Project is to design, implement, validate and apply a new flow cytometer that integrates collection of complete fluorescence emission spectra with the ability to measure and discriminate fluorescence lifetime. As described in Introduction to the application and in the Significance section below, this project directly addresses the NIH Roadmap in several areas through expanding the screening and multiplexing capabilites of flow cytometry. Our general approach will be construct the integrated instrument based on our existing phase- sensitive (PS) and high-resolution spectral (HRS) cytometery platforms. This will be accomplished through a series of four evolving aims: 1) extend the capabilities of the current instruments and data systems;2) develop and validate an integrated instrument;3) extend instrumental capabilities using region-encoding optical methods;and 4) further extend the phase- spectral range, sensitivity and resolution using swept approaches to optical encoding. We will also integrate and further expand the capabilities of the ORCA digital data acquisition and control system developed previously to handle the increasingly complex data coming from the evolving instruments. Each Aim incorporates the use of bead- and cell-based calibration and sensitivity standards for validating the performance of the evolving designs. One of the advantages of this evolving approach is that we will have new instruments available at every stage for use by collaborators, allowing both short-term applications as well as more complex uses requiring increased sensitivity, range, resolution and multiplexing capability. We have established four key collaborations in the areas of: protein engineering and screening;development and application of new cellular labels;and improved detection and measurement of intrinsic optical spectroscopy of tissues for cancer diagnosis. We anticipate many other collaborative projects once the various instruments are operational and moved into the User facility of the NFCR. Several of the developments in this third Project will also be very beneficial for the other two R&D projects in the application, including improved range and resolution of lifetime and spectral measurements (Project 1) and improved spectral resolution and CCD data handling routines (Project 2).